Suppression Of Sporulation Research Articles

CpSmt3, an ortholog of small ubiquitin-like modifier, is essential for growth, organelle function, virulence, and antiviral defense in Cryphonectria parasitica.

SUMOylation is an important post-translational modification that regulates the expression, localization, and activity of substrate proteins, thereby participating in various important cellular processes such as the cell cycle, cell metabolism, gene transcription, and antiviral activity. However, the function of SUMOylation in phytopathogenic fungi has not yet been adequately explored. A comprehensive analysis composed of proteomics, affinity pull-down, molecular and cellular approaches was performed to explore the roles of SUMOylation in Cryphonectria parasitica, the fungal pathogen responsible for chestnut blight. CpSmt3, the gene encoding the SUMO protein CpSmt3 in C. parasitica was identified and characterized. Deletion of the CpSmt3 gene resulted in defects in mycelial growth and hyphal morphology, suppression of sporulation, attenuation of virulence, weakening of stress tolerance, and elevated accumulation of hypovirus dsRNA. The ΔCpSmt3 deletion mutant exhibited an increase in mitochondrial ROS, swollen mitochondria, excess autophagy, and thickened cell walls. About 500 putative SUMO substrate proteins were identified by affinity pull-down, among which many were implicated in the cell cycle, ribosome, translation, and virulence. Proteomics and SUMO substrate analyses further revealed that deletion of CpSmt3 reduced the accumulation of CpRho1, an important protein that is involved in TOR signal transduction. Silencing of CpRho1 resulted in a phenotype similar to that of ΔCpSmt3, while overexpression of CpRho1 could partly rescue some of the prominent defects in ΔCpSmt3. Together, these findings demonstrate that SUMOylation by CpSmt3 is vitally important and provide new insights into the SUMOylation-related regulatory mechanisms in C. parasitica.

Impact of high salinity and the compatible solute glycine betaine on gene expression of Bacillus subtilis

The Gram-positive bacterium Bacillus subtilis is frequently exposed to hyperosmotic conditions. In addition to the induction of genes involved in the accumulation of compatible solutes, high salinity exerts widespread effects on B. subtilis physiology, including changes in cell wall metabolism, induction of an iron limitation response, reduced motility and suppression of sporulation. We performed a combined whole-transcriptome and proteome analysis of B. subtilis 168 cells continuously cultivated at low or high (1.2 M NaCl) salinity. Our study revealed significant changes in the expression of more than one-fourth of the protein-coding genes and of numerous non-coding RNAs. New aspects in understanding the impact of high salinity on B. subtilis include a sustained low-level induction of the SigB-dependent general stress response and strong repression of biofilm formation under high-salinity conditions. The accumulation of compatible solutes such as glycine betaine aids the cells to cope with water stress by maintaining physiologically adequate levels of turgor and also affects multiple cellular processes through interactions with cellular components. Therefore, we additionally analysed the global effects of glycine betaine on the transcriptome and proteome of B. subtilis and revealed that it influences gene expression not only under high-salinity, but also under standard growth conditions.

The Sprout Regulating Compound 1,4-Dimethylnaphthalene Exhibits Fungistatic Activity

The compound 1,4-dimethylnaphthalene, originally isolated from dormant potatoes, is currently in use as a commercial sprout inhibitor. Growers and processors report a reduction in fungal infections in potatoes treated with DMN resulting in increased yields. To assess the effects of DMN on fungal growth a culture of Fusarium oxysporum was isolated from potato tubers and identified via DNA fingerprinting using the 18ITS ribosomal region. Growth of F. oxysporum was inhibited by 31% after four days of exposure to DMN but overall rate of spore germination was not affected by DMN treatment. The growth of additional fungi, including Alternaria alternata, Aspergillus niger, Epicoccum nigrum, Gnomoniopsis smithogilvyi, Phoma medicaginis, and Pythium ultimum was inhibited by DMN as was suppression of sporulation in A. niger. These results suggest that DMN is fungistatic at the application levels examined.

Effect of light on in vivo urediniospore germination, lesion development and sporulation of Puccinia hemerocallidis on daylily and Puccinia pelargoniizonalis on geranium

The presence of rusts of daylily and geranium caused respectively by Puccinia hemerocallidis and P. pelargoniizonalis can result in reduced value of these ornamental crops. Experiments were conducted to determine the effects of fluorescent light and sunlight on urediniospore germination, germ tube elongation, lesion development and sporulation of the two fungal pathogens on detached leaves and whole plants. Exposure of dry or hydrated urediniospores of P. hemerocallidis to cool white fluorescent light (600 μmol s−1 m−2) or to sunlight (950–1910 μmol s−1 m−2) for 2 h or 4 h significantly reduced germination and germ tube elongation on detached daylily leaves. Germination but not germ tube elongation of hydrated urediniospores of P. pelargoniizonalis on detached geranium leaves was significantly reduced when exposed to fluorescent light for 2 h or 4 h. A 4 h exposure to either light source significantly reduced lesion development of P. hemerocallidis on detached daylily leaves with fewer lesions developing from hydrated compared to dry urediniospores. Sunlight exposures of 1 h and 2 h of hydrated and dry urediniospores respectively significantly reduced lesion development by either fungus on whole plants. Increasing exposure to fluorescent light negatively affected sporulation of P. hemerocallidis and P. pelargoniizonalis. Complete suppression of sporulation was not observed for either fungus with up to a 24 h exposure to fluorescent light. Light exposure negatively affected disease development by P. hemerocallidis and P. pelargoniizonalis. Exposure to high light intensities may affect spread of rust diseases on ornamental plants.

Potential of Ulocladium atrum for biocontrol of onion leaf spot through suppression of sporulation of Botrytis spp.

Epidemics of onion leaf spotcaused by Botrytis spp. depend onnecrotic leaf tissue for inoculum build up inthe crop. Ulocladium atrum Preuss. is astrong competitor on necrotic above-groundplant tissues. The potential of the antagonistto reduce colonisation of necrotic leaf tissueby Botrytis spp. and subsequentsporulation was studied in two fieldexperiments. U. atrum colonised necrotictissues and consistently reduced thesporulation of fungal competitors. Althoughincidence of Botrytis spp. was low,significantly lower spore loads of Botrytis spp. were found on spore trapslocated within U. atrum treated onionplots as compared to untreated control plots.The number of leaf spots caused by Botrytis spp. was low in both fieldexperiments but was significantly reduced by60% after U. atrum applications in thesecond experiment. Spraying of the fungicideRonilan resulted in the same control level.Results show that U. atrum has apotential for biological control against diseases caused by Botrytis spp. in onions.

Effect of the medium composition and cultivation conditions on sporulation in chemolithotrophic bacteria

The possibility of controlling endospore formation by changing cultivation conditions was for the first time shown in acidophilic chemolithotrophic bacteria Sulfobacillus thermosulfidooxidans type strain 1269 and the thermotolerant strain K1 formerly described as "S. thermosulfidooxidans subsp. thermotolerans". Suppression of sporulation occurred when these strains were cultured in Manning's liquid medium with yeast extract. This medium was optimized by gradually reducing the concentrations of ferrous iron salts (the source of energy), phosphorous, nitrogen, and yeast extract and simultaneously increasing the concentrations of calcium, magnesium, and manganese (the elements important for sporogenesis) to attain higher yields of endospores by strains 1269 and K1. As a result, a new medium A was proposed, in which the life cycle of the strains studied culminated in sporulation at a level of 45 and 60%, respectively, of the total cell number. In a series of additional tests, the growth temperature and medium pH were adjusted to obtain the maximum yield of endospores. The optimal ranges found were 40-50 degrees C and pH 1.8-2.2 for strain 1269 and 35-40 degrees C and pH 2.5-2.7 for strain K1. An even higher yield of endospores, amounting to 55 and 75% for strains 1269 and K1, respectively, was obtained when the above growth conditions were combined (growth on medium A at optimal temperatures and pH). Our results suggest a new approach to optimizing sporulation by acidophilic chemolithotrophs, which consists in limiting the energy and nutrient sources and using temperature and pH values within the tolerance bounds of these cultures but outside their growth of optimum ranges.

Effect of Nutrition and Physical Factors on Mycelial Growth and Production of Pigments and Nonchromatic UV‐absorbing Compounds ofAlternaria eichhorniae

Alternaria eichhorniaeis a host‐specific biocontrol agent for managing waterhyacinth (Eichhornia crassipes) in Egypt. An important diagnostic characteristic of this fungus is the production of crimson‐red phytotoxic pigments in the medium under certain conditions. A virulent isolate,A. eichhorniae5 (Ae5), was studied to determine the optimum conditions for its growth and production of pigments and nonchromatic UV‐absorbing metabolites (UVACs). The maximum production of pigments was obtained when cultures were grown on potato dextrose agar (PDA) with 20% dextrose, an initial pH of 4.5 at 25–30°C under continuous darkness or diurnal light, and without wrapping the culture plates. The maximum yields of the nonchromatic, 229‐ and 286‐nm‐absorbing compounds were obtained on PDA with 20–50% of dextrose at 20–30°C under continuous darkness. Culture plates, unwrapped or wrapped with only one layer of Parafilm at pH from 3.8 to 6.2 were favourable in this respect. There was a strong inverse relationship between linear mycelial growth and pigmentation as a function of light. Furthermore, the reduction in aeration, presumably proportional to the number of layers of Parafilm wrappings, led to lower levels of the red pigment(s) and the nonpigmented UVACs, to reduced mycelial growth, and a suppression of sporulation. Different culture‐filtrate fractions of Ae5 were tested for phytotoxicity. At the concentrations of 0.5 and 1% w/v of the partially purified culture filtrate fractions, all except the butanol 1 fraction, had no toxic effect on waterhyacinth leaf‐segments. Butanol fractions at 10% (w/v) concentration were significantly more damaging than aqueous fractions (10% w/v) and the area of necrosis increased with time.

Suppression of sporulation of Botrytis cinerea by antagonists applied after infection

The ability of the antagonists Epicoccum nigrum, Gliocladium roseum and Trichoderma harzianum to reduce sporulation of Botrytis cinerea when applied after infection was investigated in bean-leaf-disc assays under a variety of environmental conditions. G. roseum significantly reduced sporulation of B. cinerea when applied up to 120 h after the pathogen, whereas E. nigrum and T. harzianum suppressed sporulation when applied up to 72 h after infection. When applied 48 h after infection, all antagonists reduced sporulation of B. cinerea at temperatures of 15–25°C. G. roseum was highly effective at reducing sporulation of B. cinerea throughout this temperature range, but not at 10°C, whereas E. nigrum and T. harzianum showed their greatest relative effects on sporulation at 25°C. Antagonism by G. roseum was favoured by relative humidity of 98%, while the effect of relative humidity on E. nigrum and T. harzianum was equivocal. Under the conditions of the assay system, antagonist concentration did not have a consistent effect on reduction of sporulation.

Antifungal Properties of Some Components of Senna Alata Flower

Antifungal activity of some components of Senna alata flower was examined against five fungi. The methanolic crude extract and the partially purified fractions were both active against standard strains of Aspergillus niger, Geotricum candidum, and Candida utilis, and local isolates of Aspergillus brevipes and Penicillium species, but at different concentrations. The minimum inhibitory concentration of the methanolic crude extract was low for all the fungal strains except Aspergillus niger with 5.0 mg/mL concentration, while the minimum inhibitory concentration of partially purified components (C and F) ranged between 0.312 mg/mL for extract F against Penicillium species and 2.5 mg/mL for extract C against Aspergillus niger, Penicillium species and Candida utilis. The minimum fungicidal concentration of the crude methanolic extract was generally higher than 5 mg/mL for most of the fungi but the partially purified extract exhibited fungicidal concentrations ranging from 2.5 mg/mL to 5.0 mg/mL. Antifungal properties against mycelial growth was also demonstrated by all the extracts against the tested strains, with the crude methanolic extract losing its activity after 48 hours. The partially purified Senna alata extracts exhibited a relatively high antifungal activity against mycelial growth with total suppression of sporulation for four days at a concentration of 2 mg/mL, while preventing fungal growth after the seventh day.

Interactions between spore morphogenetic mutations affect cell types, sporulation, and pathogenesis in Magnaporthe grisea.

We have previously defined four single-gene mutations, con1, con2, con4, and con7, that control various stages of spore morphogenesis in the rice blast fungus. To delineate the developmental pathway of spore morphogenesis, we investigated the interactions among these morphogenetic genes by generating strains with double mutations via transformation-mediated gene disruption. Plasmids containing portions of the inactivated CON4 and CON7 genes were introduced into strains harboring single mutation to produce double mutants. Interaction between con1 and con4 resulted in reduced vegetative growth and suppression of sporulation. Interaction between con1 and con7 suppressed spore production but not vegetative growth. The con2/con4 and con4/con7 double mutants produced hybrid spore types with characteristic features of both parental mutants. The con2/con7 mutant produced con2 type spores, indicating that con2 is epistatic to con7 in spore morphogenesis. The epistatic relationship, however, reversed when traits related to pathogenesis were considered. Double mutants harboring the con7 mutation could not form appressoria or colonize plant tissue, indicating that con7 is epistatic to con2 and con4 in appressorium formation and pathogenesis. Thus, morphogenetic genes interact at multiple levels leading to different epistatic relationships in the pathways of spore morphogenesis, appressorium formation, and pathogenesis.

Expression analysis of the ssgA gene product, associated with sporulation and cell division in Streptomyces griseus

The ssgA gene of Streptomyces griseus B2682, when present in high copy number, results in both suppression of sporulation and fragmented growth of mycelia. Western analysis with polyclonal antibodies against the gene product (SsgA) revealed a close correlation between SsgA accumulation and the onset of sporulation in wild-type cells. The protein was only detected in the cytoplasm. Certain developmental mutants of S. griseus (afs, reIC and brgA) which are defective in aerial mycelium formation in solid culture and submerged spore formation in liquid culture failed to accumulate SsgA. The SsgA protein appeared shortly (1 h) after nutritional shift-down of strain B2682 cells. afs mutant cells sporulated and expressed SsgA only when A-factor was present both before and after nutritional shift-down. Introduction of the ssgA gene in a low-copy-number vector into strain B2682 resulted in fivefold overexpression of SsgA, and was accompanied by fragmented growth of mycelia and suppression of submerged spore formation (in liquid culture) and aerial mycelium formation (in solid culture). Streptomycin production was not inhibited. In a control experiment, a nonfunctional ssgA gene possessing a frameshift mutation near its N-terminus had no effect on either growth or sporulation. It is proposed that the ssgA gene product plays a role in promoting the developmental process of S. griseus.

Suppression of sporulation ofBotrytis spp. as a valid biocontrol strategy

In this study, the hypothesis was tested that removal of substrate for sporulation ofBotrytis spp. may lead to a retardation of an epidemic if the majority of the inoculum is produced inside the treated crop. Suppression of sporulation ofBotrytis spp. could be an attractive option for biological control ofBotrytis leaf spot in onions. In a field experiment, necrotic leaf tissue was removed to simulate the effect of a biocontrol agent. By this means, the amount of substrate on whichBotrytis spp. sporulates was reduced. In the experiment, the spore load above the onion plots was significantly reduced and the epidemic of onion leaf spot was retarded. At the end of the growing season, the number of leaf lesions in the green leaf area was lower in plots with substrate removal than in control plots (0.6 and 1.1 cm−2, respectively). The results demonstrated that an epidemic of onion leaf spot largely depends on the rate of inoculum production inside a crop. Thus, suppression of sporulation on necrotic leaf tissue is a valid control strategy that could be applied by using sporulation suppressing antagonists.

Cloning and Characterization of a Gene Involved in Regulation of Sporulation and Cell Division of Streptomyces griseus.

A mutant of Streptomyces griseus, NY5, differs from the parent in sporulating when grown in a nutrient rich growth medium. Cloned genes of the wildtype organism in the multicopy plasmid pIJ702 transformed into the NY5 mutant cells resulted in converting the mutant phenotype of sporulating in rich medium to the parental genotype of not sporulating in rich medium. A 1.5 kb DNA fragment was sequenced and found to contain two open reading frame gene regions. Restriction deletion mapping and subcloning revealed a gene which when transformed in high copy number into S. griseus wildtype, mutant NY5 and S. lividans resulted in suppression of sporulation and fragmented cell growth. The gene, named ssgA (DDJB/EMBL/GenBank accession no. is D50051), encodes a 145 amino acid protein with a calculated size of 15.8 kDa. The predicted protein has a strong negative charge and shows no significant sequence homology to known proteins. Southern analysis detected regions in S. coelicolor and S. lividans DNA homologous to ssgA.

Temperature shifts and sporulation of Bacillus megaterium

Summary: The permissive temperature for sporulation of Bacillus megaterium 27 (up to 42 °C) was found to be 4–5 °C lower than that for growth. The non-permissive temperature suppressed the initial phases of sporulation characterized by the synthesis of an extracellular proteinase but the cells retained the ability to sporulate for several hours. Neither growth at supraoptimal temperatures nor heat shock applied at the end of the growth phase increased the permissive sporulation temperature. The organism synthesized at least ten heat-shock proteins, the dominant one being HSP 69. These proteins were also found in cells after 3 h of incubation at 43·5 °C but their presence did not ensure the ability to sporulate at this temperature. The rise of temperature provoked an imbalance between synthesis and degradation of cellular proteins, whose role in suppression of sporulation is discussed.

Sporulation of Streptomyces Venezuelae in Submerged Cultures

Shaken cultures of Streptomyces venezuelae ISP5230 in minimal medium with galactose and ammonium sulphate as carbon and nitrogen sources, respectively, showed extensive sporulation after 72 h incubation at 37 degrees C. The spores formed in these cultures resembled aerial spores in their characteristics. The ability of the spores to withstand lysozyme treatment was used to monitor the progress of sporulation in cultures and to determine the physiological requirements for sporulation. In media containing ammonium sulphate as the nitrogen source, galactose was the best of six carbon sources tested. With galactose S. venezuelae ISP5230 sporulated when supplied with any of several nitrogen sources; however, an excess of nitrogen source was inhibitory. In cultures containing galactose and ammonium sulphate, sporulation was suppressed by a peptone supplement. The onset of sporulation was accompanied by a drop in intracellular GTP content. When decoyinine, an inhibitor of GMP synthase, was added to a medium containing starch and ammonium sulphate, a slight increase in sporulation was seen after 2 d. The suppression of sporulation by peptone in liquid or agar cultures was not reversed by addition of decoyinine. A hypersporulating mutant of S. venezuelae ISP5230 was altered in its ability to assimilate sugars. In cultures containing glucose the mutant sporulated more profusely than did the wild-type and did not acidify the medium to the same extent. However, the suppressive effect of glucose on sporulation was not merely a secondary result of acid accumulation.

Initiation of meiosis and sporulation in Saccharomyces cerevisiae does not require a decrease in cyclic AMP.

Meiosis and sporulation of Saccharomyces cerevisiae are initiated in a guanine auxotroph by guanine deprivation (E. Bautz Freese, Z. Olempska-Beer, A. Hartig, and E. Freese, Dev. Biol. 102:438-451, 1984). We used this condition to examine a hypothesis (K. Matsumoto, I. Uno, and T. Ishikawa, Cell 32:417-423, 1983) that initiation of meiosis requires a low level of cAMP. We found that, after guanine deprivation, the intracellular concentration of cAMP transiently decreased not more than 20% and not at all if the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) was added to the medium. Under these conditions, at least 76% of the cells sporulated in the absence of IBMX, and almost 100% sporulated in its presence. The sporulating cells continually excreted cAMP and utilized the gluconeogenic carbon source. The cells failed to sporulate efficiently and to form four-spored asci if simultaneously deprived of guanine and carbon. After guanine deprivation in glucose medium, sporulation remained suppressed and intracellular cAMP was unchanged. We conclude that, under conditions of guanine starvation, cAMP deficiency is not required for initiation of meiosis and sporulation, cAMP is produced in excess and excreted to the medium, the cells sporulate better if the cAMP concentration is increased by addition of IBMX, the cells require a gluconeogenic carbon source for complete and efficient sporulation, and suppression of sporulation by glucose is not mediated by cAMP.

Initiation of meiosis and sporulation in Saccharomyces cerevisiae does not require a decrease in cyclic AMP.

Meiosis and sporulation of Saccharomyces cerevisiae are initiated in a guanine auxotroph by guanine deprivation (E. Bautz Freese, Z. Olempska-Beer, A. Hartig, and E. Freese, Dev. Biol. 102:438-451, 1984). We used this condition to examine a hypothesis (K. Matsumoto, I. Uno, and T. Ishikawa, Cell 32:417-423, 1983) that initiation of meiosis requires a low level of cAMP. We found that, after guanine deprivation, the intracellular concentration of cAMP transiently decreased not more than 20% and not at all if the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) was added to the medium. Under these conditions, at least 76% of the cells sporulated in the absence of IBMX, and almost 100% sporulated in its presence. The sporulating cells continually excreted cAMP and utilized the gluconeogenic carbon source. The cells failed to sporulate efficiently and to form four-spored asci if simultaneously deprived of guanine and carbon. After guanine deprivation in glucose medium, sporulation remained suppressed and intracellular cAMP was unchanged. We conclude that, under conditions of guanine starvation, cAMP deficiency is not required for initiation of meiosis and sporulation, cAMP is produced in excess and excreted to the medium, the cells sporulate better if the cAMP concentration is increased by addition of IBMX, the cells require a gluconeogenic carbon source for complete and efficient sporulation, and suppression of sporulation by glucose is not mediated by cAMP.

A Forest Soil Suppressive to Phytophthora cinnamomi and Conducive to Phytophthora cryptogea. II. Suppression of Sporulation.

Extracts of soil from a wet sclerophyll eucalypt forest suppressed the formation of Phytophthora cinnamomi sporangia but facilitated production of P. cryptogea sporangia and zoospores. This suppressive action was shown to be associated in part with the microbial components of the extract and in part with the soluble components. The microbial population of the suppressive soil contained significantly greater numbers of actinomycetes than the conducive soil tested at the same time. All Streptomyes isolates from the suppressive soil were shown to be antagonistic to certain of the aerobic bacteria from the same soil and to directly inhibit mycelial growth of P. cinnamomi. The soluble component of the soil extract inhibited growth of P. cinnamomi to a greater extent than P. Cryptogea. Interactions between soil microorganisms and inhibitory compounds in the soil are considered to be the cause of the suppressive action of the soil against P. cinnamomi whilst facilitating sporulation of P. Cryptogea.

Blue-light Inhibition of Sporulation in Botrytis cinerea

Summary: Prolonged blue-light irradiation was necessary for almost total reversal of sporulation in Botrytis cinerea Pers. ex Fr. Studies of blue-light exposures of various durations during the dark period after photoinduction showed that there were two phases of maximum sensitivity to blue light, namely between 12 and 16 h, and between 20 and 24 h after the end of photoinduction. A brief exposure of 15 min to blue light (250 µW/cm2) at the 12th hour after the end of photoinduction brought about 20 % suppression of sporulation. The inhibition response was saturated at the 60 % inhibition level with more than 6 h blue-light irradiation. Blue light arrested spore formation and then caused de-differentiation of conidiophores to sterile mycelia.

Effects of hydrogen ion concentration on Phytophthora palmivora from Piper nigrum

Three-month old pepper cuttings in nutrient solutions of pH 3·0–8·0 were inoculated with Phytophthora palmivora (Butl.) Bud. on triturated pepper roots. Death occurred after 13 days at pH 6·0, 16 days at pH 8·0 and 23 days at pH 4·5. No infection occurred at pH 3·0. In the majority of twenty isolates of the causal organism kept in Petri's solution of pH 3·0–8·0, no sporangia were produced at pH 3·0, some sporangia were produced at pH 4·5, maximum sporulation occurred at pH 6·0 and declined at pH 8·0. It is suggested that disease development is prevented at low pH due to suppression of sporulation and sporangial germination.